Signals mediated by the T cell antigen receptor (TCR) complex regulate both T cell development and the balance between activation and tolerance in both early T cells (thymocytes) and mature T cells. Understanding how TCR signals regulate these processes will be essential for understanding autoimmune diseases and designing effective vaccines to pathogens. The antigen recognition ability of T cells is supplied by clonotypic chains (TCRa,b), but the signaling potential is conferred by the CD3 subunits (CD3e, CD3g,and CD3d) and members of the zeta (z)-chain family (z, eta, and Fcg). The ability of z-chain and CD3 components to transduce signals derives from a consensus sequence (ITAM) present within their intracytoplasmic domains. Each CD3 chain contains a single ITAM, whereas full length z-chain contains three ITAMs. To understand better the role of specific and multiple signaling motifs in immune processes, we have studied genetically altered mice lacking TCR-z chain and/or CD3e. We have reconstituted these mice with transgenes encoding natural or genetically engineered variants of z-chain and/or CD3e. We have used these animals to assess the role of TCR signaling in T cell development and activation. Using a breeding process in which we introduced clonotypic TCRab genes with defined specificities into signaling altered mice, we have demonstrated several key findings. 1)Thymocytes expressing TCRs with low affinity for ligand were dependent on multiple ITAM motifs for development. Importantly, TCR's with higher affinity for ligand were less dependent on multiple motifs for their development, revealing an important relationship between TCR affinity and signaling potential for development of the TCR repertoire. 2) The phenotype of cytokine responses (Th1 vs Th2) correlated with the signaling potential of the TCR. 3)The biochemical signals mediated by z chain and CD3 components are at least partially redundant. 4)The ITAM present in the CD3e chain does not have a specific function in development but rather contributes to TCR function through signal amplification. We are currently constructing mice that lack both CD3e and z-chain and will reconstitute these animals with CD3e and z-chain variant molecules to further investigate the multiple signaling structure of the TCR. Despite the dramatic effects alteration in TCR ITAMs have on T cell development, those T cells that populate the peripheral lymphoid organs in ITAM-deficient mice appear functional and proliferate in response to anti-TCR antibodies, mitogens and specific peptides. We think that expression of costimulatory molecules serve to dampen effects of deficiencies in TCR ITAM-mediated signals. Hence, we will test if costimulatory moleucles are particularly important for responses by T cells from ITAM deficient mice. We have begun a series of experiments, using purified MHC molecules loaded with specific antigen to look at the role of such costimulatory molecules in situations in which TCR signals are impaired. Finally, during the last year, we have used our model to examine how alterations in TCR signaling potential affect the development of autoimmune disease. We are using the multi-dose streptozoticin model of diabetes to determine how specific alterations in TCR signaling affect the development and pathology of diabetes. Early data suggest the signaling potential (number of ITAMs) affects the development of diabetes. Current studies are directed at understanding the inflammatory process that underlies diabetes in these mice.